s2fieldsofstudy: Biology Chemistry

text:
Multifunctional stimuli-responsive chemogenetic platform for conditional multicolor cell-selective labeling

Multicolor conditional labeling is a powerful tool that can simultaneously and selectively visualize multiple targets for bioimaging analysis of complex biological processes and cellular features. We herein report a multifunctional stimuli-responsive Fluorescence-Activating and absorption-Shifting Tag (srFAST) chemogenetic platform for multicolor cell-selective labeling. This platform comprises stimuli-responsive fluorogenic ligands and the organelle-localizable FAST. The physicochemical properties of the srFAST ligands can be tailored by modifying the optical-tunable hydroxyl group with diverse reactive groups, and their chemical decaging process caused by cell-specific stimuli induces a conditionally activatable fluorescent labeling upon binding with the FAST. Thus, the resulting switch-on srFASTs were designed for on-demand labeling of cells of interest by spatiotemporally precise photo-stimulation or unique cellular feature-dependent activation, including specific endogenous metabolites or enzyme profiles. Furthermore, diverse enzyme-activatable srFAST ligands with distinct colors were constructed and simultaneously exploited for multicolor cell-selective labeling, which allow discriminating and orthogonal labeling of three different cell types with the same protein tag. Our method provides a promising strategy for designing a stimuli-responsive chemogenetic labeling platform via facile molecular engineering of the synthetic ligands, which has great potential for conditional multicolor cell-selective labeling and cellular heterogeneity evaluation.

Introduction
Elucidating the localization and the activities of intracellular biomolecules for a deep understanding of biological processes in cells and organisms calls for effective imaging tools. [1][2][3][4] Chemogenetic strategy, as a powerful toolkit, provides many options and great potential for visualizing biological events in real-time and in situ. In this strategy, genetically encoded activators (consisting of peptides or proteins) can specically activate small synthetic ligands (uorogens) to a uorescent state by transient or covalent binding. [5][6][7] Such hybrid approaches combine the excellent targeting-specicity of the genetically encoded protein tags with superior photophysical properties as well as tunability of the small synthetic ligands, which ensures a great deal of experimental versatility and designability. 6 Accordingly, SNAP-tags, 8,9 HaloTags, 10 PYP-tags, 11,12 uorogen-activating proteins (FAPs) 13 and uorescence-activating and absorption-shiing Tags (FASTs) [14][15][16] have been developed.
As an emerging member of chemogenetic strategy, the FAST system contains a 14 kDa protein tag, which can non-covalently bind with a series of cell-permeant and nontoxic 4-hydroxybenzylidene rhodanine (HBR) derivatives and activate their uorescence. [14][15][16] The small size of the tag and its instantaneous and reversible binding with the ligands endow the FAST system with additional excellent properties such as (a) minimal functional perturbation to the target proteins, (b) excellent photophysical properties such as brightness and antiphotobleaching, and (c) much shorter maturation time than GFP-like uorescent proteins. 14 By changing the substituents on the 4-hydroxybenzyl moiety of the ligands, colours of the FAST:uorogen complex have spanned from green-yellow to farred, signicantly improving its spectral tunability. 15 Although the uorogen promiscuity of the FAST system facilitates spectral exibility, it is still challenging for FASTs to simultaneously label multiple targets with different colors because of indiscriminate recognition of structurally similar ligands by its protein tag. To address this problem, directed evolution of the FAST presented a feasible strategy to develop orthogonal labeling systems. Accordingly, two FAST variants that can selectively bind with different colored ligands (HMBR or HBR- Cite this: Chem. Sci., 2022, 13, 12187 All publication charges for this article have been paid for by the Royal Society of Chemistry 3,5DOM) were recently developed, allowing for selective labeling of two targets with dual colors. 17 However, further expanding orthogonal FAST tagging proteins requires laborious procedures to implement protein evolution and sophisticated multiple gene expressions. Hence, these challenges may present an opportunity for seeking additional solutions toward this problem.
Besides manipulation of FASTs, ligand engineering is an alternative approach with the potential to achieve multicolor selective labeling based on the identical FAST, which is yet to be demonstrated. Rationally designed ligands capable of responding to particular targets independently 18-25 may offer an opportunity to realize not only conditional selective labeling, but also multicolor selective labeling. Given that complex biological samples include many diverse cell types, cellular heterogeneity in phenotypes and genotypes, a fundamental property of cellular ecosystems, 26-28 might function as a key factor to enable conditional multicolor labeling of specic cell types of interest. The unique spatiotemporal distribution, metabolite prole, and endogenous enzymes of a cell of interest not only play a central relevance to its biological function, but are also highly useful as identication markers for discriminating diverse cell types. [28][29][30][31] It is enlightening that these cellular characteristics may act as specic triggers for stimuli-responsive ligands with different colors to achieve heterogeneity-dependent cell-selective labeling, providing an additional approach for on-demand multicolor labeling of specic cell types without the requirement of diverse protein tags.
Herein, taking advantage of the cellular heterogeneity features and rational tunability of the ligands, we introduce a FAST-derived strategy to construct a multifunctional stimuliresponsive labeling platform, namely stimuli-responsive FAST (srFAST), for conditional multicolor cell-selective labeling. The srFAST consists of two parts: one is the FAST scaffold, which can activate the uorescence of ligands exquisitely and provide an excellent subcellular location through genetic fusion with a localizable peptide or protein; the other is the rationally designed HBR-derived "caged" ligands, which are endowed with responsiveness to diverse stimuli such as light-inducible spatiotemporal activation, unique small-molecule metabolites, or the enzymatic expressions of specic cell types. These stimuli-activatable ligands can be conditionally activated by cell-specic physical or biochemical characteristics to enable on-demand uorescent labeling of FAST-fused proteins, only in target cells. Meanwhile, using multicolor enzyme-responsive srFASTs, different cell types were selectively labeled with orthogonal multicolor uorescence dependent on their independent endogenous enzymatic signatures. We envision that this proposed platform would enrich the applications of the FAST system in cell-specic multicolor labeling and provide a meaningful tool for cellular heterogeneous studies.

Results and discussion
Design principle of the srFAST platform As an emerging uorogenic chemogenetic protein tag, the FAST system features signicant uorescent enhancement and large absorption redshi of its HBR-derived ligands upon binding with the FAST, the protein ( Fig. 1a and S1 †). 14,15 These noticeable spectroscopic changes depend on the inhibition of the intramolecular rotation of ligands by FAST binding and the protein microenvironment-caused deprotonation of the ligands' phenolic hydroxyl group. In addition to the key role in the spectroscopic variation, its characteristic hydroxyl group is likely to function as an optical tunable group ready for modication-mediated regulation, which has been widely exploited in various uorescent dyes 32-35 but still unexplored in the FAST system. Accordingly, we sought to investigate whether caging the hydroxyl group of the ligands can modulate the optical properties of the FAST system.
Red-emissive ligand HBR-3,5DOM and its methoxy derivative HBR-3,4,5TOM were prepared for comparison and synthesized in one step by condensation of rhodanine to the corresponding substituted 4-hydroxy-benzaldehyde. We rst investigated the photophysical properties of the compounds ( Fig. 1b and c). The maximal absorption peak of HBR-3,4,5TOM is 393 nm, a slight blue shi to that of HBR-3,5DOM in the phenolic form (409 nm), and both compounds have little uorescence in PBS buffer, with relative uorescence quantum yields of 0.05% and 0.11%, respectively. In the presence of the FAST, deprotonated HBR-3,5DOM was stabilized and showed a large absorption redshi (409 nm to 518 nm) and a signicant uorescence increase at 600 nm. The FAST:HBR-3,5DOM complex (the original FAST system, O-FAST) exhibits a relative uorescence quantum yield of 32% (Table S1 †) comparable to that reported (31%) in the literature. 15 However, HBR-3,4,5TOM exhibited a negligible absorption change, and only a weak uorescence upon FAST binding with a relative uorescence quantum yield of 4.4%, which was about 6.3% of that of the O-FAST at 600 nm. Time-dependent density functional theory (TDDFT) calculations revealed that the protonation and methylation of the oxygen atom increase the excitation energy gaps from 2.53 (HBR-3,5DOM in phenolate form) to 2.82 (HBR-3,5DOM in phenolic form) and 2.92 eV (HBR-3,4,5TOM), respectively, supporting the similarly blue-shied absorption of the FAST:HBR-3,4,5TOM complex and free HBR-3,5DOM in comparison with the O-FAST. A close examination of the S 1 excited state (Fig. S2 †) showed that the electrons on the LUMO of HBR-3,4,5TOM and HBR-3,5DOM in the phenolic form are mainly located on the rhodanine framework, plausibly reasoning the decrease in the uorescence efficiency of the FAST:HBR-3,4,5TOM complex due to intramolecular charge transfer (ICT). 32,35 Further uorescence competition assay (Fig. S3 †) showed that HBR-3,4,5TOM could moderately interfere with the association between HBR-3,5DOM and the FAST when its concentration is signicantly higher. The proposed K d (28.2 mM) of HBR-3,4,5TOM upon FAST binding was much greater than that of HBR-3,5DOM (0.97 mM), 15 indicating a decrease in affinity. Molecular docking studies (Fig. 1d) demonstrated that HBR-3,4,5TOM could enter the binding cavity of the FAST and be stabilized by the hydrogen bond with T98 as well as apolar interactions with I31, F62, V66, A67, T70, I96, P97 and V122. However, the hydrogen-bond network formed by the conserved residues Y42 and E46 to stabilize the phenolate anion 36,37 in the O-FAST disappeared in the FAST:HBR-3,4,5TOM complex, potentially resulting in a decrease in affinity. Taken together, modifying the hydroxyl group of the ligand could diminish its binding with the FAST and efficiently reduce the complex uorescence. Hence, the feature of the optically tunable hydroxyl group provides a good entrance to designing activatable ligands for a versatile srFAST platform.
Photo-srFAST for spatiotemporal cell-selective labeling of the nucleus Light activation as an external regulation and control means has been widely used in spatiotemporal controlled imaging due to its quick, straightforward, and precisely controllable properties. 38,39 Here, an optically controlled tagging system is explored as the preliminary attempt to achieve spatiotemporally selective labeling of cells. Here, the photoactivatable ligand photo-DOM containing a 2-nitrobenzyl group 39,40 was successfully synthesized through a one-step nucleophilic reaction of HBR-3,5DOM and 1-(bromomethyl)-2-nitrobenzene (Fig. 2a). In comparison with HBR-3,5DOM, photo-DOM has a greater absorption enhancement with a large redshi from 445 nm to 567 nm aer binding with the FAST (Fig. 2b), but shows weak uorescence at 600 nm (Fig. 2c). Further competition assay indicated that 2-nitrobenzyl group modication of photo-DOM partially diminished the binding affinity with the FAST (K d ¼ 7.4 mM, Fig. S4 †). Under UV irradiation (l ¼ 365 nm, 3 mW cm −2 ), the uorescence intensity of the FAST:photo-DOM complex at 600 nm increased rapidly, and tended to be stable aer 12 min with a signicant enhancement about 15 times (Fig. 2c, d, and S5 †), which are consistent with the uncaging dynamics of the same caging group reported in other literatures. 40,41 These results proved the feasibility of our photo-responsive FAST system, named photo-srFAST. To demonstrate the ability of the photo-srFAST for spatiotemporally selective labeling, we performed patterned imaging in a 96-well plate. As shown in  , the photo-srFAST could pattern a specic "X" area with red uorescence aer UV irradiation. However for the O-FAST, red uorescence was detected in all areas without selectivity. Thus, photo-decaging of the ligand's spectrum-tunable hydroxyl group is an efficient approach to develop srFASTs, and the constructed photo-srFAST can efficiently respond to photostimulation with an excellent S/N ratio.
Then, we evaluated the feasibility of photo-srFAST in spatiotemporally selective labeling of target cells (Fig. 3a). The nucleus-localized FAST fusion protein (NLS-sfGFP-FAST) was transiently expressed in HEK293T cells, where the nuclear localization signal (NLS) and super-folded green uorescent protein (sfGFP) were fused at FAST's N-terminal sequentially. As can be seen from Fig. 3b, the nucleus of the cells emitted the typical green uorescence of sfGFP under the excitation of 488 nm, which conrmed the successful expression and localization of the FAST fusion protein. Being incubated with photo-DOM did not induce any detectable signal. Aer being illuminated with UV (l ¼ 365 nm, 3 mW cm −2 ) for 4 min, the cells appeared with red uorescence, which was colocalized well with the green uorescence, and the uorescence intensity was signicantly enhanced about 12 times aer irradiation of 10 min (Fig. 3b, c and S6 †), indicating the gradual formation of the photo-decaged uorogenic complex. Flow cytometry analysis (Fig. S7 †) also conrmed that photo-DOM could light up the target cell populations aer UV illumination. Thus, photo-DOM is cell-permeant with a negligible uorescence background, and the photo-srFAST is suitable for selective labeling of cells with a rapid and signicant uorescence response under UV irradiation. Comparatively, in the cells incubated with HBR-3,5DOM (the original FAST ligand) (Fig. 3b and c), both red and green uorescence remained bright before and aer UV irradiation, and the red uorescence from the O-FAST was bleached by 15% aer 10 min UV irradiation (Fig. S8 †). In contrast, only the cells located at the selected area exposed to UV irradiation showed red uorescence that merged as yellow in the presence of photo-DOM, while other cells out of the irradiated area remained green ( Fig. 3d and S9 †), suggesting spatially selective cell labeling by the photo-srFAST. At the same time, sequential labeling of the selected cells (Fig. 3e) was performed on a Leica DMi8 inverted microscope with an optical manipulative module, where the time and area of illumination could be nely controlled by its laser (l ¼ 405 nm, 40 mW cm −2 ). When the cells at specic locations were exposed to the laser in sequence, red uorescence appeared instantaneously (5 s) and sequentially with 8 or 11-fold uorescence enhancement, respectively, allowing for the near real-time implementation of the cell-specic labeling processes. Notably, even the adjacent cells in 50 mm can be manipulated precisely. CCK8 assay (Fig. S10 †) showed that neither photo-DOM nor illumination had an obvious inuence on the cell survival rate under the experimental conditions. Taken together, the photo-srFAST enables us to selectively and quickly label cells with high spatiotemporal imaging resolution through controlling UV irradiation, which has a potential application in spatiotemporal labeling and analysis. To demonstrate the versatility and expansibility of our srFAST platform, we sought to fabricate another stimuli-responsive ligand that can be specically activated by a particular metabolite for cell-selective labeling. Generally, different cell types respond distinctively to the uctuating environment and external stimuli and produce different metabolites under certain stress, leading to heterogeneous cellular responses. 26,27 For example, respiratory burst is a rapid release process of reactive oxygen species (ROS) usually observed in immune cells, such as macrophages, as a pathogen-defending mechanism. Phorbol 12-myristate 13-acetate (PMA) is a potent stimulant of respiratory burst of macrophages, enabling activation of NADH oxidase and consequently massive generation of hydrogen peroxide and other ROS. Recently, mounting evidence supported that H 2 O 2 produced by respiratory burst has a profound effect on macrophage signaling, 42 (Fig. S11 †), and the resulting FAST:H 2 O 2 -DOM complex exhibited relative weak uorescence at 600 nm in comparison with the O-FAST at an equimolecular concentration (Fig. S12 †). Furthermore, the competition assay between H 2 O 2 -DOM and HBR-3,5DOM revealed a moderate affinity between H 2 O 2 -DOM and the FAST (K d ¼ 5.0 mM, Fig. S13 †). Addition of H 2 O 2 triggered a remarkable enhancement in uorescence intensity due to H 2 O 2 -mediated boronate decaging of H 2 O 2 -DOM (Fig. 4b, c and S14 †). A titration experiment presented that the emission intensity of the H 2 O 2 -srFAST at 600 nm exhibited a linear response toward H 2 O 2 in the range of 5-250 mM with the detection limit of 1.5 mM (Fig. 4c and S14a †), which ts the endogenous H 2 O 2 concentration in macrophages under PMA stimulation. 42 Other compounds except for H 2 O 2 generated negligible uorescence in PBS buffer, illustrating the high selectivity of the H 2 O 2 -srFAST (Fig. S14b †).
Next, we investigated whether the H 2 O 2 -srFAST can be used for H 2 O 2 -responsive labeling of cells. HEK293T cells expressing the mitochondrial-targeted FAST protein (mito-FAST, the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase was fused at FAST's N-terminal) were treated with H 2 O 2 -DOM and a commercialized mito-tracker (a specic dye for the localization of mitochondria in living cells). Aer the addition of 200 mM H 2 O 2 , bright red uorescence was observed in cells, which overlapped with the green uorescence from the mito-tracker (Fig. S15 †) Fig. 4d, in the absence of PMA, cells incubated with H 2 O 2 -DOM showed negligible red uorescence. Aer PMA stimulation, bright red uorescence was detected, which was perfectly colocalized with the green uorescence from the mito-tracker. Flow cytometry analysis (Fig. S16 †) also depicted that the red uorescence intensity from the cell populations stimulated by PMA is about 4 times of that without PMA treatment. By contrast, the cells incubated with HBR-3,5DOM maintained red uorescence before and aer PMA stimulation. In addition, CCK8 assay suggested that H 2 O 2 -DOM had no obvious toxic or adverse effects on cells under experimental conditions (Fig. S17 †) (Fig. S18 †). Both cells expressing mito-FAST were co-cultured and stimulated with PMA, and then incubated with H 2 O 2 -DOM or HBR-3,5-DOM. For the O-FAST, it is impossible to distinguish the signals between the two different cell lines, since they were both labeled with similar red uorescence ( Fig. 4e-g). The merged images suggested the much higher transfection efficiency of HEK293T cells than RAW 264.7 cells. Even then we can easily distinguish the two co-cultured cell lines with the H 2 O 2 -srFAST. Only RAW 264.7 cells were labeled with strong red uorescence due to the high level of respiratory burst-caused endogenous H 2 O 2 , with the signal intensity more than 2 times that from HEK293T cells (Fig. 4e-g and S19 †). Therefore, the H 2 O 2 -srFAST can distinguish the cellular response of different cell types and selectively label the cell line of interest with the featured metabolite, demonstrating that our proposed srFAST platform has extensive potential in studying specic cell populations with metabolic heterogeneity.
Multicolor enzyme-srFASTs for protein-dependent orthogonal labeling of multiple cells Encouraged by cell-selective labeling based on light-triggered spatiotemporal activation and specic metabolite-dependent chemical decaging, we further adapted the srFAST platform relying on cellular heterogeneity at the expression level of endogenous enzymes. Unique cell types have cell-specic endogenous enzyme expression and activity proles to maintain diverse cellular functions, presenting a fundamental mechanism of cellular differentiation and heterogeneity. Moreover, aberrant expression of crucial enzymes in growth and metastasis regulation is highly associated with cancer development. 28 For instance, alkaline phosphatase (ALP), a hydrolase in phosphate metabolism and associated with cell differentiation and viability, is overexpressed in several tumor cell lines including HeLa cells (a cell line of cervical cancer). 44-46 b-Galactosidase (b-gal) removes galactose residues from substrates such as gangliosides, glycoproteins, and sphingolipids. It is known as an important biomarker for cell senescence and primary ovarian cancers, and is overexpressed in SKOV-3 cells (a cell line of ovarian cancer). [47][48][49] Comparatively, both enzymes are expressed at a low level in normal cells, such as HEK293T cells. 50,51 Thus, these cell-specic endogenous enzymes can be exploited to discriminate distinct cell types. We attempted to design two enzyme-activatable ligands with different colors, termed enzyme-srFASTs, for orthogonal multicolor selective labeling of cells with a distinct enzyme expression.
For responding to ALP, a ligand ALP-HMBR was designed by blocking the hydroxyl group of the green-emissive ligand HMBR with a phosphate group (Fig. 5a). The FAST:ALP-HMBR complex (ALP-srFAST) displayed very weak uorescence at 540 nm ( Fig. S20 and S21 †) and their interaction affinity was moderate (K d ¼ 5.4 mM, Fig. S22 †). As expected, the phosphate group of ALP-HMBR could be specically hydrolyzed to generate HMBR when ALP was presented (Fig. S23 †), 45,46,50 resulting in the recovery of green uorescence of the decaged FAST:HMBR complex (Fig. S24 †). The emission intensity of the ALP-srFAST at 540 nm is linearly proportional to the ALP concentration in the range of 2.0-50.0 U L −1 (Fig. S24 †). Similarly, we developed the ligand b-gal-DOM by modifying HBR-3,5DOM with a galactose group and obtained a b-gal-srFAST (Fig. 5b). Unlike other activatable HBR-3,5DOM derivatives, b-gal-DOM has no detectable change in the absorption and uorescence spectra before and aer FAST addition (Fig. S26 †). Ligand competition experiments revealed that the FAST was able to bind b-gal-DOM with low affinity (K d ¼ 16.3 mM, Fig. S27 †). Addition of b-gal caused a remarkable uorescence recovery of the FAST:b-gal-DOM complex at 600 nm owing to the hydrolyzation of the galactose group from b-gal-DOM, and there was an obvious linear relationship with the b-gal concentration from 0.01 to 0.12 U mL −1 (Fig. S28 and S29a †). [47][48][49]51 Thanks to the excellent substrate specicity, both enzyme-srFASTs exhibited good selectivity toward their targets, including the modied ligands, respectively (Fig. S24d, S25 and S29b †). Collectively, srFASTs responding to two enzymes with distinct colors were developed in vitro.
For live-cell labeling, good biocompatibility of ALP-HMBR and b-gal-DOM was rstly conrmed for their negligible inuence on cell viability in CCK8 assays ( Fig. S30 and S31 †). Next, the feasibility of the ALP-srFAST for efficient uorogenic labeling of the cells with highly expressed endogenous ALP was conrmed. Both cell imaging and ow cytometry experiments exhibited that HeLa cells expressing the membrane-localized FAST (mem-FAST, the membrane localization signal GAP43 was fused at FAST's N-terminal) appeared with typical green uorescence of the FAST:HMBR complex on their cell membrane when treated with ALP-HMBR, while there was no obvious uorescence in the cells pre-treated with Na 3 VO 4 , a typical ALP inhibitor (Fig. S32 †). Likewise, the cytomembrane of SKOV-3 cells overexpressing b-gal was labeled by the b-gal-srFAST with red uorescence, conrmed by cell imaging and ow cytometric analysis (Fig. S33 †). Together, these results proved that endogenous enzymes are potent markers for responsive labeling of target cells.
Further, the labeling selectivity of the enzyme-srFASTs was evaluated. Here, both HeLa and SKOV-3 cells transfected with the pcDNA3.1-mem-fast were separately incubated with the mixed ligands, where the uncaged ligands were devoted to verifying the successful expression and localization of mem-FAST. For the ALP-srFAST group (Fig. 5c and d), green uorescence was only observed in ALP-positive HeLa cells, and the average uorescence intensity was 6 times that of SKOV-3 cells, suggesting that the ALP-srFAST can optionally label HeLa cells. For the b-gal-srFAST group (Fig. 5e and f), only b-gal-positive SKOV-3 cells were stained by red uorescence, with the average uorescence intensity 8 times that of HeLa cells. However, for the corresponding O-FAST group, both cell lines incubated with the mixed original ligands were indiscriminately labeled with the same green or red uorescence (Fig. 5c-f). In general, the two-color enzyme-srFASTs could respond to multiplex enzymes and label different cancer cells with distinct colors, opening novel prospects for multiple cell line discrimination and analysis.
Finally, we sought to study the potential of multicolor enzyme-srFASTs for simultaneous orthogonal labeling of distinct cell lines in a complex multi-cell co-culture system (Fig. 6a). According to the different expression levels of the two enzymes, two cancer cell lines, HeLa (ALP + , b-gal − ) and SKOV-3 (ALP − , b-gal + ), and one normal cell line HEK293T (ALP − , b-gal − ) were chosen. The three types of cells expressing the same mem-FAST were co-cultured, and then incubated with the mixture of ALP-HMBR and b-gal-DOM (srFAST) or HBR-3,5DOM and HMBR (O-FAST). For the srFAST group, it is quite easy to distinguish the three cell types, as HeLa cells were labeled with green uorescence and SKOV-3 cells were labeled with red uorescence, whereas no obvious uorescence appeared in HEK293T cells (Fig. 6b-d and S34 †). For the O-FAST group, comparatively, all cells were labeled with green and red uorescence, merging as yellow equally (Fig. 6b, e, f and S34 †). Therefore, multicolor enzyme-srFASTs are suitable for selectively labeling cells with orthogonal colors via the identical protein tag, and distinguishing different cell types. The ability to respond to multiple targets by using the same protein tag but different target-responding ligands with various spectral properties provides unprecedented experimental versatility and exibility for multicolor imaging, without the need for reengineering. It is reasonable to envision that if there are cell lines co-overexpressing the two enzymes, they could be observed in yellow by employing multicolor enzyme-srFASTs. Specic labeling and distinguishing three or four cell lines with only one protein tag is a promising step toward overcoming the uorogen promiscuity of the original FAST system, which also opens up exciting prospects for conditional multicolor cell-selective labeling and cellular heterogeneity monitoring at multiplex levels.

Conclusions
In summary, we developed a multifunctional stimuli-responsive chemogenetic platform (srFAST platform) for conditional cellselective labeling via facile ligand engineering of the modular FAST system. The srFAST platform combines the subcellular localization of the genetically encoded FAST and the color and target tunability of synthetic ligands, providing excellent experimental versatility. Based on the srFAST platform, cells of interest were labeled on demand by spatiotemporally precise photo-activation or specic endogenous metabolites or enzyme proles, demonstrated by proof-of-concept models, including photo-srFAST, H 2 O 2 -srFAST or enzyme-srFASTs, respectively. Further, multicolor enzyme-srFASTs were constructed for concurrent orthogonal labeling of three cell types with different colors, proving feasibility to achieve multiplex bioimaging with the same protein tag. The generality and modularity of this platform make it facile to enrich the srFAST platform via endowing an increasing number of spectrally tunable FAST uorogens with additional responsiveness to diverse molecular features of cell heterogeneity. Moreover, the fast binding and reversible dynamics of the FAST system might enable further development of phase-specic enzyme-dependent srFAST systems to track diverse phases and progression of the cell cycle by simply changing the corresponding ligands. As a promising alternative approach for orthogonal multicolor labeling, we believe that the srFAST platform could expand the FAST toolbox and be of great potential in sophisticated imaging applications in multicellular systems.

Data availability
Experimental procedures, compound characterization, NMR spectra, and additional data are located within the ESI. †


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
CMV reactivation initiates long-term expansion and differentiation of the NK cell repertoire

Introduction NK cells play an important role in suppression of viral replication and are critical for effective control of persistent infections such as herpesviruses. Cytomegalovirus infection is associated with expansion of ‘adaptive-memory’ NK cells with a characteristic CD56dimCD16bright NKG2C+ phenotype but the mechanisms by which this population is maintained remain uncertain. Methods We studied NK cell reconstitution in patients undergoing haemopoietic stem cell transplantation and related this to CMV reactivation. Results NK cells expanded in the early post-transplant period but then remained stable in the absence of viral reactivation. However, CMV reactivation led to a rapid and sustained 10-fold increase in NK cell number. The proportion of NKG2C-expressing cells increases on all NK subsets although the kinetics of expansion peaked at 6 months on immature CD56bright cells whilst continuing to rise on the mature CD56dim pool. Phenotypic maturation was observed by acquisition of CD57 expression. Effective control of viral reactivation was seen when the peripheral NK cell count reached 20,000/ml. Discussion These data show that short term CMV reactivation acts to reprogramme hemopoiesis to drive a sustained modulation and expansion of the NK cell pool and reveal further insight into long term regulation of the innate immune repertoire by infectious challenge.

Introduction
Natural killer (NK) cells have the capacity to mediate lysis of virally-infected and transformed cells through integration of signalling from activating and inhibitory receptors (1,2) and play an important role in the control of herpesvirus infections (3). Cytomegalovirus (CMV) is not cleared after initial infection but establishes a persistent infection which acts to shape the subsequent peripheral NK cell repertoire (4). In particular, CMV-seropositive people develop expansion of a population of 'adaptive NK cells' with a predominant CD56 dim CD16 bright NKG2C+CD57+ phenotype (5, 6). This may be driven largely by engagement of NKG2C with viral peptides from the UL40 protein presented by HLA-E on virally infected cells (7) although such populations are also observed in people with an NKG2C-/-genotype (8). The NKG2 proteins are important regulators of NK activation and both NKG2A and NKG2C form a heterodimer with CD94 that binds to HLA-E (9,10). However, whilst NKG2A acts as an inhibitory receptor and is predominantly a marker of immature NK cells, NKG2C is an activating receptor that is expressed on highly differentiated populations. The potential functional importance of this adaptive NK cell subset remains somewhat unclear although it has high capacity for antibody-dependent cellular cytotoxicity (ADCC).
Allogeneic haemopoietic stem cell transplantation (HSCT) is an important therapeutic modality within haemato-oncology and has the potential to cure patients with chemotherapyresistant disease (11). Much of this curative effect is mediated through the activity of the donor immune system (12) and there is increasing interest in the potential role of NK cells in this regard. The number of NK cells within the donor graft correlates with protection from disease relapse after allo-HSCT (13) and early NK reconstitution can help to suppress GvHD whilst increasing the 'graft versus leukaemia' effect (14)(15)(16). Patients are highly immune suppressed in the early period after HSCT and reactivation of CMV is an important clinical complication that is associated with increased rates of morbidity and mortality (17,18). However, despite the increased short term clinical risk mediated by CMV reactivation it has also been demonstrated that viral reactivation may reduce subsequent post-transplant leukaemia relapse (19)(20)(21). The mechanism behind this observation remains uncertain but may relate to the generation of high numbers of CMV-driven donor NKG2C+ NK cells that increase capacity for control of host tumour cells.
Although CMV reactivation after hempoietic or solid organ transplantation has been shown to boost the number of mature NKG2C+ NK cells (22,23) relatively little is known regarding the kinetics of this response from the time of reactivation. Here we determined the profile peripheral NK cell repertoire in patients after an episode of CMV reactivation following HSCT. Importantly, samples were taken immediately after viral reactivation and then followed prospectively. We observe that CMV viral reactivation leads to a long term expansion of NKG2C+ NK cells that transitions across the NK differentiation repertoire over a period of at least 10 months. Transient viral reactivation is thus seen to reprogramme long term NK cell differentiation and reveals further insights into pathogen-driven regulation of lymphopoiesis.
2 Materials and methods 2.1 Patients and control subjects 41 patients who had undergone allo-HSCT in the Queen Elizabeth Hospital were recruited at the time of CMV reactivation following written informed consent under the BOOST ethics (RG 13-172) according to Declaration of Helsinki. 60ml of peripheral blood in sodium heparin (BD Vacutainer ® , Cat#367876) was collected for the first four weeks following CMV reactivation and 36ml of heparinised and clotted blood was also available pre-transplant, prereactivation and for up to 10 months post-transplant. Samples were also collected from patients undergoing allo-HSCT but without CMV reactivation (n=25) at equivalent time points preand post-transplant for use as controls. This group of patients were also followed for up to 10 months post-transplant.
All 66 patients who underwent allo-HSCT with a 10/10 HLAmatched sibling or unrelated donor were recruited to this prospective study (Table 1) and PCR monitoring for CMV reactivation performed weekly. 12/66 (18%) patients received myeloablative transplants, 7 using myeloablative total body irradiation plus chemotherapy, and 5 chemotherapy alone. The remaining 82% (54/66) received reduced intensity chemotherapy conditioning. 61 patients (92%) received T cell depletion, with approximately half receiving ATG (n=28) or alemtuzumab (n=31), whilst 2 patients received post-transplant cyclophosphamide as part of a haploidentical transplant. All patients received ciclosporin based GvhD prophylaxis, with methotrexate added to myeloblative regimens, and mycophopheolate mofetil to all busulphan containing regimens. All patients received PBSC-mobilised stem cell grafts. Approximately one third of patients (n=20) had a matched related (sibling) donor, whilst two patients had a mismatched related haplo-identical donor. Almost two thirds of patients (n=40) had a matched (10/10) unrelated donor, and one had a 9/10 mismatched unrelated donor. The remaining 3 patients received double umbilical cord transplants. These baseline characteristics of patients are summarised in Table 1 and stratified according to the presence of subsequent CMV reactivation.

Assessment of CMV serostatus and CMV reactivation
Patients were routinely monitored for CMV reactivation by quantitative PCR and reactivation was diagnosed by value of >200 copies/ml of CMV DNA in whole blood. CMV viral load was monitored for each week following reactivation. Patients were classified according to their CMV serostatus pair; recipient positive and donor positive (R+/D+); recipient positive and donor negative (R+/D-); recipient negative and donor positive (R-/D+) and recipient negative and donor negative (R-/D-). Donors with ambiguous CMV serostatus are classified as equivocal (R+/Dequi).
All patients received anti-viral propylaxis with acyclovir. Patients with a matched unrelated donor received IV aciclovir 500mg/m 2 tds until mucositis resolved and then 800mg qds orally until day +100. Those who received a stem cell graft from a sibling donor were given oral aciclovir 200mg qds until day +35. Following viral reactivation patients received valganciclovir when viral load was >10,000 copies/ml.   --1254). Up to 60 ml of peripheral blood was collected in sodium heparin tubes and CMV serostatus was determined.

Isolation of peripheral blood mononuclear cells
PBMCs were isolated by density gradient centrifugation over Lymphoprep ™ (Stem cell Technologies, Cat #07861) within 24 hours of collection. Peripheral blood was diluted 1:1 with RPMI-1640 Medium (Sigma, Cat#R8758) with 1% Penicillin/Streptomycin (Gibco ™ Cat#15070063), layered at a ratio of 2:1 and centrifuged at 2000 x g for 25 minutes (brake off). Buffy coats were harvested and resuspended in RPMI media and centrifuged at 500 x g for 10 minutes and the number of cells determined using a haemocytometer (Fast-Read 102 ® , Kova International, Cat #88010). Cells were washed and centrifuged at 500 x g for 10 minutes before being cryopreserved in freezing media (RPMI-1640 Medium [Sigma, Cat#R8758], 10% DMSO [Sigma, Cat #D2650]).

Intracellular cytokines staining
one million PBMCs from CMV reactivation, without reactivation and healthy donors were co-cultured with K562 cells overnight (1:1 ratio) in incubator and suspended within 500 µl of growth media with of Brefeldin A (Sigma, Cat #B5936). The cells were spun down, washed next day. Surface markers were stained before the cells were fixed and permeabilized. At last anti-human TNF-a-AF488 [BioLegend, Cat#502917], antihuman IFN-g-AF700 [BioLegend,Cat#502519] were added, incubated and washed. The data was collected from the Gallios ™ and analysed using Kaluza ® Flow Analysis software version 2.1a (Beckman Coulter Inc.).

Absolute NK cell count
The absolute NK cell count was calculated by applying the percentage of NK cells, according the flow cytometry staining (gated from the live and lymphocyte subsets, Supplementary  Figure 1), to the total lymphocyte count (10 9 /L) obtained from the clinical haematology laboratory.

Statistical analysis
Data were analysed by using Mann-Whitney test for comparison between patients' groups and Wilcoxon sign rank test for different time-points within similar patients. Friedman test was used for comparison in more than two groups within similar group of patients. All sample sizes in the different timepoints are small and treated as not normally distributed and analysed by using GraphPad Prism 8 and SPSS 20.

CMV reactivation following HSCT drives rapid and long term expansion of NK cells
66 patients who underwent allo-HSCT with a 10/10 HLAmatched sibling or unrelated donor were recruited to this prospective study (Table 1) and PCR monitoring for CMV reactivation performed weekly. These baseline characteristics of patients are summarised in Table 1 and stratified according to the presence of subsequent CMV reactivation.
CMV reactivation was observed in 41 patients (62%) at an average time of 52 days post-transplant. Blood samples were then taken at the time of reactivation and at weeks 1, 2, 3, 4, and months 3, 6, 10, after CMV reactivation. Control post-transplant samples were also taken from patients without evidence of viral reactivation and were matched for equivalent timepoints of collection. Blood samples were also available pre-transplant and pre-reactivation from almost all donors.
In initial studies, the percentage of NK cells within peripheral blood was determined using flow cytometry ( Figure 1). The proportion of NK cells was markedly higher in patients compared to healthy donors (HD) and reflects early and robust NK reconstitution in the early post-transplant setting. Indeed, NK cells comprised over 50% of PBMC within the first 4-weeks post-HSCT and prior to any episode of viral reactivation. This value remained stable following CMV reactivation before declining gradually after 4 weeks towards the normal range ( Figure 1A). Moreover, the absolute number of NK cells also increased rapidly following CMV reactivation but then continued to rise further over the next 10 months. Indeed, values rose 11-fold from 3 cells/µl at the time of reactivation to 35 cells/µl at the final timepoint of assessment at 10 months ( Figure 1C). In contrast, the NK cell number remained very stable in patients who did not experience CMV reactivation ( Figure 1D). These observations reveal that short term CMV reactivation drives rapid and long term NK cell expansion.
3.2 CD56 dim CD16 bright NK cells expressing NKG2C undergo a rapid and sustained expansion following CMV reactivation in patients after allo-HSCT CD56 dim CD16 bright NK cells comprise the dominant peripheral population of NK cells and we next went on to assess the relative expression of NKG2C and NKG2A on this subset in relation to CMV reactivation (Figure 2A). Average expression of NKG2C in healthy CMV seronegative donors was 1.4% ( ± 0.2) compared to 11% (± 3.5%) in seropositive individuals (p<0.0001). Values for NKG2A + were 50% ( ± 2.6%) and 40% ( ± 3.2%) respectively (p=0.0065) indicating that CMV-driven expansion of the NKG2C+ pool comes at the expense of a decrease in the NKG2A+ repertoire ( Figure 2B). Within CMV-seropositive HSCT patients the percentage of NKG2C + NK cells was comparable to that seen in CMVseropositive controls prior to reactivation. However, a rapid expansion of CD56 dim CD16 bright NK cells expressing NKG2C was observed within the first 7 days after viral reactivation. Furthermore, this population continued to expand for a further 10 months following clearance of viral reactivation such that the NKG2C + cell proportion represented up to 36% of the total NK cell pool, a value far higher than the normal range in CMVseropositive healthy donors ( Figure 2C). A much more modest increment was seen in patients who did not suffer an episode of CMV reactivation. The proportion of NKG2A + cells within HSCT patients was initially higher than in healthy donors and is likely to reflect production of immature NKG2A + NK cells during NK reconstitution. However, CMV reactivation accelerated NK cell maturation with an early decrease in the proportion of NKG2A + cells after reactivation and a continuing fall to~30% at month 10, a value below that seen in controls ( Figure 2C).
The NKG2C:NKG2A ratio was next determined on the NK cell pool at different time points. Within healthy donors this was very low at 0.03:1 in CMV-seronegative people compared to 0.3:1 in the CMV-seropositive group ( Figure 2D). Within HSCT patients the ratio was initially 0.1:1 but following viral reactivation it increased to reach 0.2-0.5:1 at 3 months, comparable to values seen in CMV-seropositive HD. Moreover, this increased further such that at 10 months the NKG2C+ cell percentage exceeded that of NKG2A+ with a ratio of 1.2:1. These data show that there is a rapid and long term expansion of the NKG2C+ NK cell subset following CMV reactivation in patients after allo-HSCT and that the relative proportion of this subset expands above that seen in healthy CMV seropositive people.

The proportion of NKG2C-expressing cells increases on all NK cells subsets following CMV reactivation and peaks earlier on immature populations
CMV-driven adaptive memory NK cells have been characterised previously only within the CD56 dim CD16 bright NK cell subset. The relative expression of CD16 and CD56 is a marker of NK cell differentation and, given the finding of increased NKG2C expression on the cytotoxic CD56 dim CD16 bright pool following CMV reactivation, we next went on to determine NKG2C and NKG2A expression on additional NK cell subsets ( Figure 3A).
The percentage of CD56 bright cells was increased in HSCT patients compared with controls, irrespective of CMV reactivation, and reflects accumulation of immature NK cells during early post-transplant lymphoid reconstitution ( Figure 3B). Interestingly, the proportion of NKG2C-expressing cells increases on all NK cells subsets and was associated with reciprocal loss of NKG2A + (Figures 3C, D). NKG2C expression was 3-4 fold higher on mature cytotoxic CD56 dim NK subsets compared to CD56 bright cells. In addition, it was noteworthy that the kinetics of NKG2C expression differed between these two populations with NKG2C expression peaking at 6 months (representative examples are shown on Supplementary Figure 2) on CD56 bright cells whilst continuing to increase to month 10 on CD56 dim cells.
These data indicate that CMV-driven enhancement of NKG2C expression is imprinted at the immature stage of NK differentiation, peaking at 6-months, whilst continuing to accumulate on more mature subsets for a further 4 months.

Transplantation from CMV seropositive donors modulates the profile of viraemia following reactivation
We next went on to determine how the CMV-serostatus of the haemopoietic transplant donor (D) acted to influence the magnitude of viral reactivation and profile of NK maturation following CMV reactivation in the patient ('recipient'; R). Transplantation using a CMV-seropositive donor will infuse CMV-specific immune memory populations within the stem cell graft whilst immune cells from a CMV-seronegative donor will need to develop a primary CMV-specific immune response to control reactivation. 54% of patients who suffered an episode of CMV reactivation patients had R+/D+ CMV-serostatus whilst 37% had an R+/D-profile. The magnitude and kinetics of viral load at reactivation were influenced by donor CMV serostatus. Of note, the initial viral load at the time of CMV reactivation was higher in R+/D+ patients (2350 ± 810 copies/ml vs 897 ± 270 copies/ml in R+/Dpatients; (p=0.05) ( Figure 4A). However, the subsequent peak viral load was considerably higher in the R+/D-cohort, indicating a role for transferred CMV-specific immunity in accelerating control of viral replication. Two or more episodes of viraemia were seen in 79% R+/D-patients (11 out of 14) but only 26% of R+/D+ patients (6 out of 23). Second peaks of viraemia occurred most commonly between weeks 2 and 4 and were typically higher than seen at initial reactivation. Viral load became virtually undetectable when the absolute NK count rose above 20/ml suggesting that this may represent a potential biomarker for effective control of viraemia.
A trend towards a differential pattern of expression of NKG2C and NKG2A was observed in relation to donor serostatus ( Figures 4B, C) but the number and percentage of NK cells were not altered and no difference was observed in extended NK cell phenotype ( Supplementary Figures 3 and 4).
These data suggest that transfer of memory NKG2C+ NK cells from a CMV seropositive donor may contribute to control of viraemia although little impact is seen on NK cell phenotype or number after reactivation.

NK cells show a more activated phenotype and produce higher cytokine levels after HCMV reactivation in HSCT patients
The pattern of expression of the early activation marker CD69 and the differentiation-associated protein CD57 was then assessed on the cytotoxic CD56 dim CD16 bright NK subset. Of note, the percentage of CD69 + NK cells was much higher in HSCT patients compared to healthy donors at virtually all time points after transplant and is likely to reflect an influence of homeostatic proliferation ( Figure 5A). The highest level of expression was seen at the time of CMV reactivation and then fell over the next few weeks whilst values were stable in the absence of reactivation. Interestingly, no significant difference in CD69 expression among four NK subsets (NKG2A-/ NKG2C-, NKG2A+/NKG2C-, NKG2A-/NKG2C+, NKG2A +/NKG2C+) was seen at any time point (Supplementary Figure 6). The proportion of CD57 + NK cells decreased rapidly after CMV reactivation and remained low until week 4 before returning to the normal range at 3 months and increasing further thereafter ( Figure 5A). In contrast, the proportion of CD57+ NK cells remained very stable in the absence of reactivation. An increase in the proportion of KIRs+ NK cells was also apparent after HCMV reactivation, most notably within the first 4 weeks, although no significant change was seen in relation to the pattern of PD-1 or KLRG1 expression (Supplementary Figure 5).
The profile of TNF-a and IFN-g production by NK cells was also determined in relation to prior history of viral reactivation. Due to limited sample availability, analysis was undertaken at 6-10 months post transplant and NK cells were co-cultured with K562 cells and assessed by intracellular flow cytometry. Higher baseline levels of cytokine production were seen post-transplant compared to controls and may reflect increased baseline proliferation as shown above ( Figure 5B). However, activationinduced cytokine production was markedly increased in patients who had experienced an episode of CMV reactivation. In particular, TNF-a and IFN-g production was observed in 61% and 73% of NK cells respectively compared to only 21% and 25% in patients without reactivation ( Figure 5B). A higher level of cytokine production was seen from CD56 dim NK cells compared with CD56 bri NK cells (Supplementary Figure 7). Furthermore, the highest level of cytokines were seen within NKG2A-/ NKG2C-NK cells followed by the NKG2A-/NKG2C+ subset, whilst very few NKG2A+/NKG2C+ NK cells were cytokine positive ( Figure 5C). Cytotoxic responses against K562 were comparable between groups (data not shown).
These findings show that an episode of CMV reactivation serves to drive NK cell proliferation and differentiation and mediates a sustained increase in capacity for inflammatory cytokine production.

Discussion
CMV establishes a persistent infection that is controlled by immune surveillance and leads to imprinting of the peripheral lymphoid repertoire (4). We find that short term viral reactivation leads to rapid and sustained expansion of NK subsets over a period of many months. Our studies were undertaken in immune suppressed patients after allogeneic stem cell transplantation but the findings are likely to inform directly on the mechanisms that control CMV reactivation within healthy immune competent people. As such they provide insight into the immune control of CMV replication and further demonstration of how pathogens can modulate the long term profile of innate immunity.
NK cells are amongst the first lymphocyte subsets to reconstitute after HSCT and this was reflected in both the high percentage of NK cells and an increased proportion of immature subsets in the early post-transplant period (16). However, an episode of CMV reactivation was seen to have a profound impact on the subsequent profile of NK cell maturation and expansion. CMV reactivation occurred at a median of 52 days which is consistent with previous reports and reflects early reactivation of virus from host tissues (22). The number of NK cells in the early post-transplant period was low but was not predictive of reactivation risk. In patients who did not experience an episode of viral reactivation the percentage and number of NK cells remained broadly stable over the next 10 months. In contrast, viral reactivation led to a significant expansion of NK cells within 3-weeks which then continued such that NK cell number increased by 11-fold after 10 months. Interestingly, this increase was accompanied by a reduction in the NK cell percentage within the peripheral repertoire and reflects the profound impact of CMV reactivation on expansion of other lymphoid subsets, most notably T cells (24). The nature of HSCT transplant conditioning and donor choice is likely to impact substantially on the profile of NK reconstitution and similar findings have been seen after umbilical donor transplantion (22)although an increase in NK number was not seen in the setting of haploidential transplantation (25,26). In the renal transplant setting a recent study has revealed a similar finding of increased number and frequency of NK cells after HCMV reactivation (27).
A particular focus was on the kinetics of the adaptive memory NKG2C+CD56 dim CD16 bright NK subset that is expanded in CMV seropositive donors. The NKG2C genotyping was not included in this study, but there are two CMV seronegative HD controls and one non CMV reactivation HSCT patient in this study do not have NKG2C+ NK cells at all. We took advantage of early sampling to show that this population increased within 2 weeks of reactivation and the proportion of NKG2C-expressing cells increased incrementally from 4% to 35% of the CD56 dim CD16 bright pool over 10 months. Expression NKG2C or NKG2A was largely mutually exclusive and a comparable fall in the percentage of the NKG2A population was therefore seen over this period. There is also increasing interest in the potential role of CD56 negative NK cells and Hess and colleagues have demonstrated expansion of CD56 neg CD16 bri NK cells in donors with CMV and EBV coinfection (28) although we were not able to study this subset within our analysis.
Previous studies have identified CMV-driven NKG2C+ adaptive memory cells only within the mature CD56 dim NK population but here we also found this to develop on CD56 bright populations. Indeed, a relative increase in proportion of NKG2C-expressing cells was seen on all four NK subsets as defined by CD56 and CD16 expression although the relative kinetics and magnitude were markedly different. The increment in NKG2C expression on the immature CD56 bright subsets was much more modest than seen on CD56 dim cells, with maximal values of 8% and 15% for the CD16 neg and CD16 bright subsets respectively. A particular finding of interest was that NKG2C + expression on CD56 bright cells peaked at 6 months whilst that on the CD56 dim subset continued to increase at month 10. CD56 bright cells are believed to be precursors of the CD56 dim population (29) and as such these data indicate that NKG2C + CD56 dim cell expansion following CMV reactivation is at least partially supported by differentiation from NKG2C+ CD56 bright NK cell precursors during the first 6-months ( Figure 6). However, studies in patients with GATA2 mutation or PNH have indicated that adaptive cells may have self-renewing properties and as such the lineage derivation of this pool remains unclear (30,31). In addition recent data from murine models has shown that an ILC1-like splenicresident memory pool is induced following murine CMV infection (32) and the broad impact of viral reactivation on the repertoire of innate memory is therefore likely to have been underestimated (33).
The functional importance of transfer of 'immune' adaptive memory NK cells from the transplant donor was assessed by comparison of immune reconstitution between transplants with either a CMV-seropositive or seronegative donor. It was noteworthy that the initial viral load was elevated following transplantation from a CMV seropositive donor and this may reflect either viral replication from donor leucocytes or a consequence of donor CMV-specific immunity acting to transiently enhance short term viral load at reactivation (34). Despite this effect, peak viral load and longer term control of viremia were improved following transplantation from a CMVseropositive donor. NK cells retain and transfer immune memory to facilitate control of viral reactivation (35) and a trend was seen towards more rapid NKG2C + cell expansion within the first four weeks after reactivation within the R+/D+ group although NK cell number and phenotype were broadly comparable. Of note, prospective analysis allowed assessment of NK number in relation to control of viral reactivation and identified that viral load was controlled in all subjects when the peripheral NK cell count reached 20,000/ml (20/ml), comparable to values of 18/ml associated with higher overall survival at day 60 post transplant (36). The lymphocyte count in peripheral blood of healthy donors is~10 6 /ml and, with an NK cell percentage of 2-10%, this number is comparable to the value of 20-100,000 NK cells/ml in CMV-seropositive healthy donors. Sustained control of viral reactivation is likely to also require T cells and the role of adaptive memory cells in antigen presentation is of note in this regard (37).
Analysis of the kinetics of the extended phenotype of CD56 dim CD16 bright NK cells after viral reactivation focussed on CD57 and CD69. Expression of CD57, a marker of late differentiation, was somewhat lower in the first month postreactivation but then increased continuously over subsequent months. As such, the classical expression of CD57 and KIR on adaptive memory NK cells is seen to develop late after reactivation (38) and supports evidence for coupling of differentiation and proliferation of NK cells over an extended period (26,39,40). It is not clear why expression was reduced during the first month after reactivation although this may reflect preferential recruitment of this subset into tissue. CD69 is upregulated upon NK cell activation (41) and expression was broadly increased within all patients which is likely to reflect in the impact of homeostatic proliferation and immune reconstitution. However, values were at their highest level at the time of viral reactivation and then gradually fell and there was no correlation between CD69 and NKG2C expression.
The relative functional capacity of NK cells was assessed by the profile of cytokine secretion and cytotoxic capacity following activation in vitro. Cytokine production was generally enhanced within NK cells from HSCT patients compared to healthy donors and may reflect the increased proliferative status in this setting. However, CMV reactivation further primed for the production of high levels of TNF-a and IFN-g, as seen previously (22), and consistent with epigenetic modification of the IFNG locus (42). There is interest in the potential beneficial Model of potential maintenance of the adaptive memory NK cell pool through reactivation of CMV within bone marrow. CMV reactivation leads to a pattern of increased peripheral expression of NKG2C on both CD56 dim mature NK cells and the more immature CD56 bright subset. The peak of this expansion occurs earlier, at 6 months, in the immature population compared to 10 months in the mature subset. Viral reactivation may potentially engage both populations independently or CD56 dim cells may differentiate from the more immature CD56 bright subset.
impact that may arise from CMV-driven enhancement of NK maturation (43) and reactivation has been associated with a reduction in the risk of leukaemia relapse in some studies although the underlying mechanisms remain unclear (44,45).
Homeostatic expansion (46, 47) and the high level of viral reactivation are both likely to enhance NK cell expansion in the post-transplant period and likely to underlie the 'supraphysiological' expansion of the NKG2C+ NK cell subsets that was observed. However, the broad features relating to the kinetics and phenotypic profile of viral-induced NK expansion are likely to be applicable to immune competent populations (48). NKG2C+ cells remain elevated for decades following CMV infection and, as hemopoietic and myeloid precursors are reservoirs for CMV latency, these observations suggest that this may be maintained by intermittent viral reactivation acting to modulate NK cell differentiation within the bone marrow. The finding that NK cell expansion is sustained for at least 10 months might appear surprising given that innate responses are typically regarded as reactive and short term. Very low levels of subclinical viral reactivation in the posttransplant setting cannot be ruled out but were not detected by sensitive PCR. Moreover, the findings concur with observations that other innate cells, such as neutrophils and monocytes, also show long term peripheral changes after acute events and suggest that epigenetic regulation of stem cell precursors acts to establish long term adaptive memory (49, 50). Limitations of our study were that we were unable to determine NKG2C genotype in participants and the cessation of sampling at 10 months after reactivation.
In conclusion we show that CMV reactivation drives rapid and long term expansion of NKG2C+ NK cells over a period of at least 10 months. Indeed, this mechanism might underlie the maintenance of expanded NKG2C+ populations within CMV seropositive healthy donors but this was not addressed in this study ( Figure 6). These findings are of potential importance for management of CMV reactivation after HSCT and suggest that NKG2C+ NK cell monitoring might be used to predict immune capacity to control reactivation. Furthermore, this profile of expansion following shortterm viral reactivation extends insight into pathogen-driven haemopoietic programming of stem cell precursors to enhance innate cell memory.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by West Midlands -South Birmingham Research Ethics Committee 'REC reference number: 15/WM/0194'. The patients/participants provided their written informed consent to participate in this study.


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Fufang Xueshuantong Improves Diabetic Cardiomyopathy by Regulating the Wnt/β-Catenin Pathway

Diabetic cardiomyopathy (DCM) is one of the main complications of diabetic patients and the major reason for the high prevalence of heart failure in diabetic patients. Fufang Xueshuantong (FXST) is a traditional Chinese medicine formula commonly used in the treatment of diabetic retinopathy and stable angina pectoris. However, the role of FXST in DCM has not yet been clarified. This study was conducted to investigate the effects of FXST on diabetic myocardial lesions and reveal its molecular mechanism. The rats were intraperitoneally injected with 65 mg/kg streptozotocin (STZ) to induce diabetes mellitus (DM). DM rats were given saline or FXST. The rats in the control group were intraperitoneally injected with an equal amount of sodium citrate buffer and gavaged with saline. After 12 weeks, echocardiography, heart weight index (HWI), and myocardial pathological changes were determined. The expression of transforming growth factor-beta1 (TGF-β1), collagen I, and collagen III was examined using immunofluorescence staining and western blot. The expressions of Wnt/β-catenin signaling pathway-related proteins and mRNA were detected by western blot and real-time PCR. The results showed that FXST significantly improved cardiac function, ameliorated histopathological changes, and decreased HWI in the DM rats. FXST significantly inhibited the expression of myocardial TGF-β1, collagen I, and collagen III in DM rats. Furthermore, FXST significantly inhibited the Wnt/β-catenin pathway. Taken together, FXST has a protective effect on DCM, which might be mediated by suppressing the Wnt/β-catenin pathway.

Introduction
Diabetic cardiomyopathy (DCM) is a major cause of the high prevalence of heart failure in diabetic patients [1]. DCM is characterized by specific myocardial structural and functional abnormalities at different stages, which cannot be completely explained by other cardiovascular or noncardiovascular reasons [2]. e main pathological mechanisms of DCM are hyperglycemia, reactive oxygen species, abnormal fatty acid metabolism, microvascular dysfunction, and myocardial fibrosis [3,4]. At present, the treatment of DCM is still based on antiglycemic agents, combined with angiotensin-converting enzyme inhibitors, diuretics, and β-blockers drugs. However, the risk of drug intolerance and electrolyte disorders must be considered [4].
erefore, further studies are needed to elucidate the pathogenesis of DCM and investigate new therapies.
Myocardial fibrosis is one of the principal pathological features in the late development of DCM [5]. Hyperglycemia usually induces abnormal proliferation of myocardial fibroblasts and deposition of extracellular matrix (ECM) to accelerate the process of myocardial fibrosis, ultimately leading to cardiac insufficiency [6,7]. It was reported that the Wnt/β-catenin signaling pathway can regulate cardiac fibroblast activation and ECM secretion during cardiac fibrosis [8,9]. In addition, the previous study showed that the protein expression of Wnt2 and β-catenin is upregulated in the left ventricles of streptozotocin (STZ)-induced diabetes mellitus (DM) rats, and blocking the Wnt/β-catenin signaling pathway can put off the development of DCM [10].
e clinical experiments demonstrated that FXST can effectively relieve traditional Chinese medicine syndromes of patients with stable angina pectoris of coronary heart disease, can cut down the dosage of nitroglycerin, and has no obvious toxic side effects [16]. But there are few reports about the effects of FXST on DCM. Hence, the purpose of this study was to observe the effects of FXST on DCM and reveal its molecular mechanism, which might provide new options for DCM patients.

FXST Preparation.
e preparation method of FXST was in accordance with the Chinese Pharmacopoeia (2020). Panax notoginseng (Burk.) F. H. Chen (250 g) was crushed and extracted twice with 50% ethanol, the filtrate was combined, the ethanol was recovered and concentrated into an ointment, and the dregs were dried and pulverized into fine powder for use. e remaining three herbs such as Astragalus membranaceus (Fisch.) Bunge (80 g), Salvia miltiorrhiza Bunge (50 g), and Scrophularia ningpoensis Hemsl. (80 g) were heated and refluxed with 50% ethanol two times. en the filtrate was combined, ethanol was recovered and concentrated to an appropriate amount, and finally mixed with the above-prepared ointment and fine powder, dried, crushed, and obtained. e ultra-performance liquid chromatography-mass spectrometry analysis of FXST was determined in our previous work [14].

Animals and Treatment.
Male Sprague-Dawley rats weighing 180-200 g were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). e rats were raised in a specific pathogen-free environment, with 12 h light/12 h dark cycles and free access to food and water. All animal experimental protocols were approved by the Ethical Committee of Experimental Animal Care, China Academy of Chinese Medical Sciences, and complied with the National Institutes of Health guidelines (Guide for the care and use of laboratory animals).
After two weeks of adaptive feeding, the rats in the control group (n � 8) were intraperitoneally injected with sodium citrate buffer. e remaining rats were injected with an equal volume of 65 mg/kg STZ dissolved in a 0.1 M sodium citrate buffer (pH 4.5) to induce diabetes [17,18]. Following one week of treatment, blood was collected from the tail vein to measure the blood glucose. If the fasting blood glucose level was higher than or equal to 16.7 mmol/L, the DM model was successfully established. Rats with blood glucose <16.7 mmol/L, were excluded from the study. e DM rats were randomly divided into 2 groups: the DM group (n = 12) and the FXST group (n = 12). e rats in the FXST group were orally gavaged with FXST (1.05 g/kg/ day) once a day for 12 weeks. e rats in the control and DM groups were orally gavaged with an equal amount of saline. e body weight and blood glucose of the rats were measured at 0, 4, 8, and 12 weeks and 1, 2, 4, 8, and 12 weeks, respectively. At the end of the experiment, the insulin level was measured using enzyme-linked immunosorbent assay analysis according to the manufacturer's instructions (80-INSRT-E01, ALPCO, USA).

Hemodynamic Detection.
After 12 weeks of drug intervention, the rats were weighed, anesthetized by inhalation with isoflurane, and fixed in the supine position. A Standard II lead electrocardiogram was monitored. e hemodynamic examination was performed according to the requirements of the MP150 multichannel physiological signal recording and analysis system (Biopac, USA). e right common carotid arteries of the rats were separated, the distal ends were ligated, and the proximal ends were clamped with an arterial clamp. A 20 G vein indwelling needle was inserted through the right carotid artery and connected with the pressure transducer, and then detected by an MP150 multiguide physiological recorder. Cardiac hemodynamic indexes were recorded: heart rate, cardiac index (CI), left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), dp/dt min, and dp/dt max.

Echocardiography.
e echocardiography was conducted using the Vevo 770 ultrasound imaging system (Visual Sonies, Canada). e rats were anesthetized and fixed, and the left ventricle long and short axis sections were obtained using an RMV-716 probe at a frequency of 17.5 MHz at the left sternal border. Ejection fraction (EF) and fractional shortening (FS) were measured and analyzed. e ultrasonic measurements were averaged over 3 consecutive cardiac cycles. Following the measurements of cardiac function, blood from the abdominal aorta of rats was collected, the whole heart was weighed, and the heart weight index (HWI � heart weight/body weight) was calculated.

Histopathologic Evaluation.
e heart tissue was fixed in 4% paraformaldehyde for 48 h, dehydrated by an ethanol gradient, and then embedded in paraffin. Paraffin-embedded heart tissues were sliced into 4 μm sections. Hematoxylineosin staining and Masson staining were performed to evaluate histological changes. e morphology of the left ventricle was observed under the microscope. Additionally, the collagen volume fraction was quantitatively analyzed by the Image-Pro-Plus 6.0 Image software.

Immunofluorescence.
e sections were routinely deparaffinized to water, followed by antigenic repair, and then nonspecific epitopes were blocked. e slices were incubated with the primary antibody (TGF-β1, collagen I, collagen III, 1 : 100 dilution) overnight at 4°C. On the following day, slides were washed with phosphate buffer saline three times and incubated with fluorescein-labeled secondary antibody for 1 h at 37°C. e nuclei were stained with 4′,6-diamidino-2-phenylindole for 5 min. e images were captured by a laser scanning confocal microscope (Leica, SP8, Germany) and analyzed for fluorescence intensity with Image J software.

Western Blot.
Prechilled RIPA lysate (p0013 B, Beyotime, Shanghai, China) with a protease inhibitor mixture was added to the myocardial tissues. e tissues were fully ground and lysed. e homogenate was allowed to stand for 10 min on ice and centrifuged at 12,000 r/min for 20 min at 4°C to obtain the protein from the supernatant. e protein concentration was detected with the protein quantification kit (MB155207 A, Pierce, USA). Subsequently, the proteins were separated on a SDS/PAGE gel and transferred to the polyvinylidene fluoride membrane (Millipore, Massachusetts, USA). e membrane was blocked with 5% skim milk and incubated with β-actin, TGF-β1, collagen I, collagen III, phospho-GSK-3β, GSK-3β, β-catenin, Wnt2, c-Myc, and WISP1 antibodies at 4°C overnight. After being washed with tris-buffered saline and tween 20, the membranes were incubated with goat anti-rabbit IgG or goat anti-mouse IgG (Sc-2004/Sc-2005, Santa Cruz Biotechnology, Inc. USA). β-actin served as the internal control. e protein bands were detected using a gel imager (Bio-Rad, USA) and quantified with Quantity One v.4.6.2 software.

Real-Time PCR.
Total RNA was extracted from cardiac tissues using Trizol reagent (15596018, Ambion, USA) according to the manufacturer's instructions. e RNA concentration was determined using a NanoDrop ND-2000 spectrophotometer ( ermo Scientific, USA). en, the RNA was reverse transcribed into cDNA using a reverse transcription kit (Ambion, USA). e real-time polymerase chain reaction was performed with the SYBR Green qPCR Master Mix on an Applied Biosystems Prism 7500 Sequence Detection System. e β-actin mRNA expression level was used as the internal control. e 2 −ΔΔCt method was used to calculate the expression of the target genes. e PCR primer sequences (5′ ⟶ 3′) were shown as follows: e data were reported as the mean ± standard deviation (SD). When the data conformed to the normal distribution, one-way analysis of variance (ANOVA) was used for comparison among multiple groups followed by the least significant difference (LSD) test for equal variances or the Games-Howell test for unequal variances; When the data did not conform to the normal distribution, Kruskal-Wallis test was used for comparison among multiple groups (SPSS software 23.0, SPSS Inc., Chicago, IL). P < 0.05 was considered statistically significant.

FXST Increased Insulin Concentration in the DM Rats.
e typical symptoms of STZ-induced type 1 diabetic rats included polydipsia, polyphagia, polyuria, emaciation, hyperglycemia, and low insulin levels [19]. As shown in Figure 1(a), the body weight of the control group rats increased steadily, while the weight of the DM group rats was significantly lower than that of the control group (P < 0.01 or 0.001), and there was no significant difference in body weight International Journal of Endocrinology between the DM and FXST group (P > 0.05). Blood glucose was significantly elevated in the DM group as compared to the control group (P < 0.01 or 0.001), and FXST treatment had no significant effect on blood glucose in the diabetic rats (P > 0.05, Figure 1(b)). In addition, insulin concentration in the DM group was significantly reduced compared to the control group (P < 0.01). e insulin concentration of the FXST group was higher than that of the DM group without a statistical difference (P > 0.05, Figure 1(c)).

FXST Improved Cardiac Function in the DM Rats.
e left ventricular hemodynamic and echocardiography parameters were measured to assess the cardiac diastolic and systolic function of the rats. As shown in Figures 2(a)-2(i), compared with the control group, the heart rate, CI, LVSP, dp/dt min, dp/dt max, EF, and FS of the DM group significantly decreased (P < 0.01 or 0.001). FXST treatment significantly increased heart rate, CI, LVSP, dp/dt min, EF, and FS (P < 0.05 or 0.01). FXST treatment exhibited a trend to enhance dp/dt max (P > 0.05). In addition, LVEDP was increased while FXST was decreased in DM rats (P > 0.05).
ese results testified that FXST could alleviate cardiac dysfunction and delay DCM progression in diabetic rats.

FXST Improved Myocardial Histomorphology and
Structure in the DM Rats. As shown in Figure 3(a), myocardial hematoxylin-eosin staining showed that cardiomyocytes were orderly arranged with uniform nuclear and cytoplasmic staining in the control group. In the DM group, cardiomyocytes were disordered with inflammatory cell infiltration, and uneven nuclear and cytoplasmic staining, which were reversed after FXST intervention. Besides, the effect of FXST was also evaluated by HWI (Figure 3(b)). Compared with the control group, the HWI of the DM group significantly increased (P < 0.001), whereas FXST markedly reduced HWI (P < 0.01). e results showed that FXST could significantly alleviate the abnormal myocardial tissue morphology and structure of diabetic rats.

Rats.
e results of Masson staining showed that the deposition of myocardial collagen fibers in the heart tissue of the DM group was significantly higher than that of the control group (P < 0.001). Inversely, the deposition area of collagen fibers in the myocardial tissue of the FXST group was significantly reduced compared with the DM group and the degree of fibrosis was reduced (P < 0.01, Figures 4(a)-4(b)). We further determined the levels of collagen I, collagen III, and TGF-β1 by immunofluorescence and Western blot. e expression levels of collagen I, collagen III, and TGF-β1 was significantly elevated in the myocardium of the DM group compared with the control group (P < 0.05 or 0.01 or 0.001), nevertheless, FXST downregulated the expression of collagen I, collagen III, and TGF-β1 compared to the DM group (P < 0.05 or 0.01 or 0.001, Figures 4(c)-4(k)). As consequence, FXST could inhibit the expression of fibrosis-associated genes in DCM.
e results of the real-time PCR determined that compared to the control group, the myocardial mRNA Wnt2, β-catenin, GSK-3β, and c-Myc in the DM group were significantly increased (P < 0.05 or 0.01 or 0.001), by contrast, the mRNA expression levels of β-catenin and c-Myc were significantly decreased in the FXST group compared with the DM group (P < 0.05 or 0.01, Figures 5(h)-5(k)).

Discussion
In the present study, we investigated the effect of FXST in STZ-induced diabetic rats and confirmed the following points. (1) FXST improved cardiac function and

FXST Improved the Cardiac Function and Pathology of DM Rats.
In the current study, we found that body weight loss, elevated blood glucose levels, HWI, cardiomyocyte disorder, and myocardial fiber gap widening were the main pathological features in STZ-induced diabetic rats. ese changes were consistent with the reports of the DCM rat model [19]. It has been reported that FXST improves the hemodynamic status in acute myocardial ischemic canines established by ligating the coronary artery anterior descending branch [20]. Panax notoginseng saponins and Salvia miltiorrhiza Bunge, the main components of FXST, increased EF, FS, and LVSP and decreased HWI in the myocardial infarction rats [21,22]. Moreover, Tanshinone IIA, one of the main monomers of Salvia miltiorrhiza Bunge, has been demonstrated to ameliorate DCM [23]. Notoginsenoside R1, a newly extracted phytoestrogen from Panax notoginseng (Burk.) F. H. Chen could suppress oxidative stress and cardiac fibrosis to prevent DCM in the db/ db mice [24]. e above findings indicated that FXST may become a potential therapeutic drug for DCM. Accordingly, we assessed the pharmacologic effects of FXST in the STZinduced DCM rats. In this study, we clarified that FXST improved the cardiac function of DCM rats. FXST enhanced EF and FS, maintained hemodynamic stabilization and decreased HWI in the STZ-induced DM rats. Furthermore, the pathological changes of myocardial tissues were also partially restored following FXST treatment. However, we found no significant difference in blood glucose levels between the DM and FXST groups. Based on the concept of "preventive treatment of disease" in Chinese medicine, if diabetic rats are pretreated with FXST in advance, it might play an imperative role in lowering blood glucose and better preventing myocardial injury. Hence, we will investigate whether FXST pretreatment could inhibit the development of STZ-induced diabetes in future studies.

FXST Mitigated Myocardial Fibrosis in the DM Rats.
Myocardial fibrosis is a key factor in the deterioration of DCM cardiac function. e characteristic of fibrosis is excessive accumulation of ECM [25], and the fibrin in the ECM is mainly comprised of collagen I and collagen III. Studies have confirmed that the reduction of collagen I and collagen III alleviates myocardial fibrosis in STZ-induced diabetic mice [26,27]. TGF-β1 activation can increase the deposition of ECM in the myocardial interstitium which consequently LVEDP, (f ) Heart rate, (g) CI, (h) dp/dt max, and (i) dp/dt min were detected. Data are expressed as the mean ± SD (n � 4-6 for each group). * * P < 0.01, * * * P < 0.001 vs. the Control group, # P < 0.05, ## P < 0.01 vs. the DM group. e heart weight index of each group was calculated. Data are expressed as the mean ± SD (n � 5 for each group). Scale bar � 50 μm. * * * P < 0.001 vs. the Control group, ## P < 0.01 vs. the DM group. 6 International Journal of Endocrinology  lead to an increase in the myocardial hardness and a decrease in the ventricular systolic and diastolic function [28]. Our data showed that in comparison with the control group, the protein expression of collagen I, collagen III, and TGF-β1 was significantly increased in the DM group, indicating that collagen I, collagen III, and TGF-β1 were involved in the progress of DCM. Additionally, studies have determined that tanshinone IIA, astragaloside, and ginsenoside Rb1, which are representative bioactive ingredients of FXST, play a crucial role in inhibiting the development of cardiac fibrosis [29][30][31]. In our study, compared to the DM group, the protein expressions of TGF-β1, collagen I and collagen III were remarkably reduced by FXST. erefore, we could assume that FXST attenuates the fibrosis process.

FXST Inhibited the Wnt/β-Catenin Pathway.
e components of the canonical Wnt pathway include ligands (Wnt), transmembrane receptors, cytoplasmic regulatory proteins (GSK-3β, β-catenin), and nuclear transcription factors. Under myocardial injury conditions, the Wnt pathway is activated. Wnt protein binds to cell surface receptors, resulting in the depolymerization of the GSK-3β/ APC/Axin degradation complex, which inhibits the phosphorylation of β-catenin by GSK-3β. Subsequently, β-catenin is accumulated in the cytoplasm and then transferred to the nucleus, thus turning on the transcription of downstream target genes (c-Myc, WISP1) [32,33].
e Wnt/ β-catenin pathway exerts an imperative role in the regulation of DCM [34] and myocardial fibrosis [9]. β-catenin protein expression is usually elevated in various cardiac fibrotic lesions [35]. GSK-3β can regulate cardiac hypertrophy and cardiac fibrosis [36]. Under stimulation factors such as ischemia and hypoxia, the dysregulation of c-Myc expression causes fibroblast proliferation and the increase of collagen synthesis, leading to myocardial fibrosis [37]. WISP1 is a member of the connective tissue growth factor family, which promotes proliferation and fibrosis [38]. In our study, the gene and protein expression levels of β-catenin, WISP1, Wnt2, c-Myc, and GSK-3β in the myocardial tissues of the DM group were increased compared to those of the control group. Besides, it was reported that the downregulation of Wnt2, GSK-3β, and β-catenin expression may ameliorate cardiac fibrosis in diabetic mice and type 1 diabetic rat  Data are expressed as the mean ± SD (n � 4 for each group). * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. the Control group, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the DM group. [39,40]. Studies have shown that the effective ingredients of FXST, tanshinone IIA, and ginsenoside Rb1, can inhibit Wnt and β-catenin levels in hyperglycemic circumstances [41,42]. In line with the literature, we found the expressions of β-catenin, WISP1, p-GSK-3β, Wnt2, and c-Myc in the FXST group were significantly inhibited. It is suggested that the therapeutic effect of FXST on DCM cardiac fibrosis is at least partially attributed to the inhibition of the Wnt/ β-catenin pathway. However, in future studies, we intend to use Wnt inhibitors or Wnt transgenic mice to more directly demonstrate the cardioprotective effect of FXST through the Wnt/β-catenin pathway.

Conclusion
In summary, FXST could enhance cardiac function and reduce myocardial damage caused by diabetes, and its antimyocardial fibrosis mechanism might be related to the downregulation of the Wnt/β-catenin signaling pathway. ese findings provide an experimental basis for understanding the beneficial role of FXST in the treatment of DCM.
e components of FXST are pretty complex, therefore further studies on the active ingredients of FXST should be carried out. In addition, in-depth research can be conducted to explore the components of FXST that can act on fibrosis and the Wnt pathway.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.


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P030 Highly slow-release antifungal wound dressing for chronic dermatophytosis

Abstract   Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives The aim of this research was to make a biocompatible and affordable nanofibrous wound dressing that is able to release terbinafine at the site of chronic superficial fungal infection over time. Methods Polymer solution (10%) of poly (caprolactone) (PCL) was prepared in hexafluoroisopropanol (HFIP). Terbinafine hydrochloride (TFH) was added equal to 5% of PCL weight for drug-loaded samples. Electrospinning was performed with a 27-G-needle equipped syringe at a distance of 15 cm which the injection flow rate of the solution was 0.2 ml/h and a 30 kV voltage was applied. The measurements of drug release were performed with HPLC. Antifungal tests were done on three different fungal species and MTT assay was done based on ISO-10993 on 24 h L929 cells. The drug release was monitored for 144 h in a human body simulated system (incubation at 37°C, shaking by 30 rpm, and passing the drug through a wet Whatman filter into PBS). ∷Ξ Results The mean diameter of fibers was obtained at 1262 nm for PCL nanofibers without TFH and 249 nm for PCL nanofibers with TFH. The drug loading for PCL was 83.4%. Antifungal activity of PCL fibers was examined against a dermatophyte (Trichophyton mentagrophytes), a saprophyte (Aspergillus fumigatus), and a yeast (Candida albicans). The terbinafine hydrochloride-contained electrospun PCL fibers inhibited the growth of T. Mentagrophytes and A. Fumigatus but did not inhibit C. albicans growth. None of the samples showed cytotoxic effects after 24 h and 2 weeks. Conclusions The diameter of PCL nanofibers with TFH apparently decreased by fives times (P >.05). PCL nanofibers successfully inhibited two important fungal species while no toxicity was observed in MTT assay for its extraction of 2 weeks. They were able to release TFH slowly over time which makes them suitable for the treatment of chronic superficial fungal infections.

Poster session 1, September 21, 2022, 12:30 PM -1:30 PM Objectives: The aim of this research was to make a biocompatible and affordable nanofibrous wound dressing that is able to release terbinafine at the site of chronic superficial fungal infection over time.
Methods: Polymer solution ( 10% ) of poly ( caprolactone ) ( PCL ) was prepared in hexafluoroisopropanol ( HFIP ) . Terbinafine hydrochloride ( TFH ) was added equal to 5% of PCL weight for drug-loaded samples. Electrospinning was performed with a 27-G-needle equipped syringe at a distance of 15 cm which the injection flow rate of the solution was 0.2 ml/h and a 30 kV voltage was applied. The measurements of drug release were performed with HPL C. Antifungal tests were done on three different fungal species and MTT assay was done based on ISO-10993 on 24 h L929 cells. The drug release was monitored for 144 h in a human body simulated system ( incubation at 37°C, shaking by 30 rpm, and passing the drug through a wet Whatman filter into PBS ) .
ࢽ Results: The mean diameter of fibers was obtained at 1262 n m for PCL nanofibers without TFH and 249 n m for PCL nanofibers with TFH. The drug loading for PCL was 83.4%. Antifungal activity of PCL fibers was examined against a dermatophyte ( Trichophyton mentagrophytes ) , a saprophyte ( Aspergillus fumigatus ) , and a yeast ( Candida albicans ) . The terbinafine hydrochloride-contained electrospun PCL fibers inhibited the growth of T. Mentagrophytes and A. Fumigatus but did not inhibit C. albicans growth. None of the samples showed cytotoxic effects after 24 h and 2 weeks.
Conclusions: The diameter of PCL nanofibers with TFH apparently decreased by fives times ( P > .05 ) . PCL nanofibers successfully inhibited two important fungal species while no toxicity was observed in MTT assay for its extraction of 2 weeks. They were able to release TFH slowly over time which makes them suitable for the treatment of chronic superficial fungal infections. Objectives: Invasive Candidiasis is a life-threatening condition that kills a large number of immunocompromised patients each year around the world. We used post-antifungal effect studies to analyze the activities of anidulafungin, as a clinically crucial antifungal drug, amphotericin B, and fluconazole ( alone and in combinations ) against FLC-susceptible and -resistant Candida albicans isolates obtained from the cancer patients.
Methods: We tested the phenomenon of post-antifungal effects ( PAFEs ) of fluconazole ( FLC ) , amphotericin B ( AMB ) , anidulafungin ( AFG ) , and combinations of FLC + AFG, AFG + AMB, and FLC + AMB against 17 C. albicans isolates obtained from the oral cavity of cancer patients. Isolates that had not been exposed to antifungals, served as a control group. Colony counts were performed at 0, 2, 4, 6, and 24 h after a brief ( 1 h ) antifungal exposure.
Results: The FLC had no detectable post-antifungal effect independent of antifungal concentration and resembled drugfree FLC ( control ) . When all AMB and AFG were compared with FLC, significant variations in the post-antifungal effect were observed. Combining AFG and AMB with FLC resulted in effective activity compared to FLC alone. Combination regimens were rated as indifferent in general. Interestingly, low dosages of the AFG displayed increasing fungistatic action as it neared a fungistatic endpoint against C. albicans isolates ( n = 17 ) .
Conclusion: Our findings suggest that brief exposure to AFG, in combination with FLC and AMB, at low concentrations of the medicines utilized, could be effective in the evaluation and optimization of new dosage regimens to manage candidiasis. However, future research will look at the clinical utility of our findings. and novel treatment strategies to combat later infections. A novel compound Aryl-1, 2, 4-triazol-3-ylthio, fluconazole alcoholderived analogs ( ATTAFs ) , has newly developed with potent in vitro activity against Candida species, including fluconazoleresistant isolates. The objective of this study was to further evaluate the in vivo effectiveness in a murine model of invasive candidiasis due to C. albicans .
Methods: Treatment with ATTAF-1 and ATTAF-2 significantly increased the survival of infected mice compared to the control group ( 5% DMSO plus inoculum ) .
Results: The antifungal action of ATTAF-1 and ATTAF-2 and their median survival time provided no evidence of a difference versus fluconazole. Although there was an obvious fungal load ( mean log CFU/g of tissue ) decrease by ATTAF-1 and ATTAF-2 in the kidney, spleen, and liver of the treated mice in comparison with the control group and not similar to each other in regard of the dose, fluconazole showed a significant decrease in the number of fungal loads, similar to the group treated with ATTAF-1 and ATTAF-2. Nevertheless, the results of this study indicate that the use of ATTAF-1 and ATTAF-2 as a therapeutic agent can exert significant in vitro and in vivo antifungal effects against C. albicans , increasing animal survival and significantly decreasing fungal loads.
Conclusion: Although we have identified two new compounds, ATTAF-1 and ATTAF-2, as novel promising Candidates for the treatment of Candida infection, more studies of ATTAF-1 and ATTAF-2 activity and their action mechanisms in animal models are warranted to enhance our understanding and establish their efficacy. Objective: The epidemic of antifungal-resistant dermatophytosis in India has been reported. These infections are associated with severe morbidity, resistance to oral itraconazole and terbinafine, and the widespread misuse of topical steroids. Trichophyton indotineae has emerged as the predominant causative agent. In this study, we investigated 162 dermatophytosis infections from eight Indian states using genomic sequencing. The primary objective was to determine whether a clonal outbreak strain is responsible for the current epidemic .
Methods: A total of 161 T. indotineae and one T. rubrum isolates from skin scrapings collected from India in 2017-2019 and previously reported were sent to the U.S. Centers for Disease Control and Prevention ( CDC ) for genomic analysis. After species identification, genomic DNA was extracted and sequenced using Illumina NovaSeq. Single-nucleotide ( SNP ) analysis was performed using the portable workflow MycoSNP ( v0.21 ) . Briefly, MycoSNP prepared the reference genome, performed pre-processing, aligned sample reads to the reference using the BWA ( v 0.7.17 ) alignment algorithm, and called variants using GATK ( v4.1.4.1 ) . High-quality SNPs were used for constructing phylogenetic trees using neighbor-joining ( NJ ) and maximum likelihood ( ML ) methods. Further, to understand if infections are genetically clustered by state or region, multi-dimensional scaling ( MDS ) was applied using the ML tree in R.
Result: SNP analysis identified 1259 450 variant sites which were used to construct an NJ and ML tree. The tree topology from both NJ and ML methods showed consensus. All 161 T. indotineae isolates from India clustered together forming a large, well-supported clade. SNP differences between the samples varied from 0-160 SNPs. Historical isolates available at CDC were included as controls and clustered over 40 000 SNPs from the clade comprising isolates from India. The MDS plot revealed that isolates did not cluster by state or region.
Conclusion: Antifungal-resistant dermatophytosis is an emerging threat with cases of chronic, recurrent infection reported from several countries including India. Additionally, the rapid spread of infections involves person-to-person spread. Our results suggest that a clonal outbreak of a T. indotineae strain is circulating in multiple states in India. Current plans are to expand the geographic scope of the study by including over 10 countries from Europe, Middle East, and the Americas. This work will allow the public health community to better understand the emergence and transmission of antifungal-resistant dermatophytosis worldwide.

Non-conventional alternatives to prevent Candida auris infections
Andra-Cristina Bostanaru, Valentin Nastasa, Carla Pavlov-Enescu, Eugen Hnatiuc, Mihai Mares Iasi University of Life Sciences, IASI, Romania Poster session 1, September 21, 2022, 12:30 PM -1:30 PM Objectives: Candida auris represents a particular organism associated with multiple nosocomial infections and high mortality rates. Appropriate infection control measures play a major role in controlling the spread and multiplication of this pathogen. Unfortunately, there are very few data available on the effectiveness of disinfectants against C. auris . Chlorine-based products appear to be the most effective for inanimate surface disinfection, while iodine-based products are more suitable for skin antisepsis. C. auris has been demonstrated to survive on dry and moist surfaces for up to 14 days, so environmental cleaning to eliminate a source of nosocomial infections is a challenge. Thus, searching for new effective fungicidal agents is still a hot topic nowadays. Non-thermal plasma-activated water ( PAW ) has recently emerged as a powerful antimicrobial agent, but no data about its effectiveness on C. auris are available. The aim of our study was to assess the possibility of using PAW as a fungicidal agent against C. auris planktonic cells.
A type strain of C. auris ( CBS 10913 ) was used in this study. Suspensions of yeast cells ( 107 CFU/ml ) were prepared from overnight cultures and subsequently treated with PAW in a ratio of 1:10 for different periods of time ( 1, 3, 5, 7, 10, 15, and 20 minutes ) . Precise volumes of the mixtures were further inoculated on Sabouraud Dextrose Agar plates and Bact/Alert FA Plus bottles ( bioMerieux, France ) in order to evaluate the reduction of yeast burden after each contact period. In addition, some instrumental analysis ( IA ) methods were used in order to assess the impact of PAW treatment on yeast cell structure: Scanning Electron Microscopy ( SEM ) , Atomic Force Microscopy ( AFM ) , Fourier Transform Infrared Spectrometry ( FT-IR ) , and Dynamic Light Scattering ( DLS ) . All tests were performed in triplicates.
Results: A reduction higher than 5 log10 of viable yeast was achieved in 3 minutes. The sterilization level ( i.e., ˃6 log10 reduction ) was achieved after 5 minutes for the tested strain. IA clearly objectified the morphological changes in the treated yeasts compared with the untreated ones ( Figs. 1 and 2 ) .
Conclusion: Our research has successfully demonstrated the fungicidal effect of PAW against C. auris , opening a new field of research in the area of disinfectants.